Tumor suppressive role of a 2.4 Mb 9q33-q34 critical region and DEC1 in esophageal squamous cell carcinoma

The key genes involved in the development of esophageal squamous cell carcinoma (ESCC) remain to be elucidated. Previous studies indicate extensive genomic alterations occur on chromosome 9 in ESCC. Using a monochromosome transfer approach, this study provides functional evidence and narrows down the critical region (CR) responsible for chromosome 9 tumor suppressing activity to a 2.4 Mb region mapping to 9q33-q34 between markers D9S1798 and D9S61. Interestingly, a high prevalence of allelic loss in this CR is also observed in primary ESCC tumors by microsatellite typing. Allelic loss is found in 30/34 (88%) tumors and the loss of heterozygosity (LOH) frequency ranges from 67 to 86%. Absent to low expression of a 9q32 candidate tumor suppressor gene (TSG), DEC1 (deleted in esophageal cancer 1), is detected in four Asian ESCC cell lines. Stably expressing DEC1 transfectants provide functional evidence for inhibition of tumor growth in nude mice and DEC1 expression is decreased in tumor segregants arising after long-term selection in vivo. There is 74% LOH in the DEC1 region of ESCC primary tumors. This study provides the first functional evidence for the presence of critical tumor suppressive regions on 9q33-q34. DEC1 is a candidate TSG that may be involved in ESCC development.     

Three distinct regions of deletion on 13q in squamous cell carcinoma of the larynx

Genetic etiology of squamous cell carcinoma of the larynx (SCCL) is very complex, with both molecular and chromosomal alterations involved. The target genes have not yet been clearly identified. Therefore, our study focused on searching for regions that potentially harbor genes related to SCCL. After comparative genomic hybridization (CGH) analysis of a set of 52 SCCL we specified 13q21-q32 and 13q34 as the most frequently deleted regions. In order to precisely map the critical region of deletion, we studied these areas by using 15 microsatellite markers. In our material a significantly high frequency of loss of heterozygosity (LOH) (test for a difference in two proportions, p<0.001) was observed for the following markers: D13S1320 (13q21.1), D13S800 (13q21.3), D13S1818 (13q32.1), D13S770 (13q32.3) and D13S285 (13q34). Three hot spots of LOH were found: 13q21.1-q22.1 (D13S1320-D13S1824-D13S800-SHGC30014- WI-16413-D13S1186), 13q.31.1-13q32.3 (D13S317-D13S1818-D13S770), 13q34 (D13S285). Among these areas, 13q31.1-q32.3 was identified as new hot spot of deletion in SCCLs.     

Identification of a minimal deletion region on chromosome 5q in Chinese esophageal squamous cell carcinomas

The existence of unknown tumor suppressor gene(s) other than the APC gene has been hinted on 5q for esophageal squamous cell carcinoma (ESCC). In order to define minimal deletion intervals on 5q in ESCC and investigate the potential tumor suppressor gene(s), 9 microsatellite markers scattering the region from 5q22 to 5q35 were chosen for loss of heterozygosity (LOH) analysis in 50 primary ESCC from northern China. The results showed that six cases presented coexistence of LOH for three consecutive adjacent chosen markers, suggesting a minimal deletion region covering approximately 272 kb located on 5q23 from D5S1384 to D5S1505. It was a novel deletion region that was so far never reported in ESCC. Significant higher frequencies of LOH were observed in tumors with lower pathological grade at the locus D5S820 and with lymph node metastasis at the locus D5S408. The data suggested the possibility that one or more putative candidate tumor suppressor gene(s) on 5q23 might play an important role in the development and/or progression of ESCC.     

Lymph node metastasis is associated with allelic loss on chromosome 13q12-13 in esophageal squamous cell carcinoma.

Allelotype analysis of whole chromosomes showed that loss of heterozygosity (LOH) on 13q was exclusively associated with lymph node metastasis and poor prognosis in esophageal squamous cell carcinoma (ESC). To identify a locus responsible for lymph node metastasis, we performed fine deletion mapping on 13q by analyzing 60 ESCs with 18 polymorphic markers. Allelic loss was observed with at least one marker in 34 tumors (56.7%), and lymph node metastasis was significantly correlated with LOH (P = 0.0053). We found frequent loss at D13S260 (43.7%), D13S171 (38.6%), and D13S267 (43.6%) on 13q12-13. Among these markers, LOH at D13S171 showed a significant correlation with lymph node metastasis (P = 0.0441). Because these markers flank the BRCA2 gene, we intensively examined a mutation of the gene through all coding exons and exon-intron junctions by PCR-single-strand conformational polymorphism analysis under two different assays. We found only seven nucleotide substitutions as normal polymorphic changes; tumor-specific mutations were not detected, and loss of expression was not observed, indicating that the BRCA2 gene might not be a target of allelic loss in this region. Relatively frequent LOH was also found at the RB1 locus (34.7%), but a significant correlation with lymph node metastasis was not observed (P = 0.7430). Protein expression of RB1 was examined in 31 ESC cell lines, and loss of expression was infrequent (6.5%), indicating that inactivation of the RB1 gene might not be responsible for lymph node metastasis. Taken together, allelic loss at 13q12-13 of the primary ESC was closely associated with lymph node metastasis, and unidentified tumor suppressor gene(s) in this region might be involved.     

Interstitial chromosomal deletion within 4q11-q13 in a human hepatoma cell line.

Southern blot analysis revealed the presence of an aberrant albumin gene as well as a normal one in a human hepatoma cell line, HuH-7. A genomic sequence carrying this altered gene was isolated and characterized. This clone contains a 3-kbp 3' segment of the albumin gene linked to a non-albumin sequence at intron 11. The non-albumin sequence is assigned to chromosome 4q12-q13 by in situ hybridization. This indicates an interstitial deletion of a chromosomal segment within 4q11-q13 because the albumin gene is mapped there. The truncated albumin gene is detected in an early passage of HuH-7 cells and has been maintained stably in cell culture.     

High-resolution deletion mapping of 15q13.2-q21.1 in transitional cell carcinoma of the bladder

Deletions found in several types of human tumor, including carcinomas of the colorectum, breast, and lung, suggest the presence of a potential tumor suppressor gene(s) on chromosome 15. Common regions of deletion in these tumors are at 15q15 and 15q21. Here, we have analyzed loss of heterozygosity (LOH) on chromosome 15 to ascertain its potential involvement in the development and progression of transitional cell carcinoma (TCC) of the bladder. A panel of 26 polymorphic markers, spanning 15q12-15q22, were used to map regions of LOH in 51 TCCs. LOH was found for at least one marker in the region 15q14-15q15.3 in 20 of 51 (39%) tumors. Deletion mapping defined two minimum regions of deletion: a distal region between the markers D15S514 and D15S537 at 15q15.1-15q15.3 (estimated as 3 Mb) and a more proximal region between the markers D15S971 and D15S1042 at 15q14 (estimated as 1.1 Mb). Analysis of a panel of 33 bladder tumor cell lines revealed regions of contiguous homozygosity for markers in 15q15, indicating likely LOH. Fluorescence in situ hybridization analysis demonstrated that mitotic recombination is the predicted mechanism of LOH in two of these. These regions of LOH on 15q may contain tumor suppressor genes the loss or inactivation of which is associated with TCC development. The DNA repair gene RAD51 at 15q15.1 represents a candidate 15q tumor suppressor gene. Expression analysis of rad51 protein in tumor cell lines revealed variable levels of expression but no significant loss of expression in cell lines with likely 15q LOH.     

Allelic loss on chromosome 13q14 and mutation in deleted in cancer 1 gene in esophageal squamous cell carcinoma

Previous studies have shown frequent allelic loss on chromosome 13 in esophageal squamous cell carcinoma (ESCC). We assessed the frequency of allelic loss on chromosome 13q14 and mutations of deleted in cancer 1 (DICE1) (also found on 13q14) in ESCC patients to determine if this candidate tumor suppressor gene has a role in the development of ESCC, and whether this gene was an inactivation target for allelic loss on chromosome 13q14. Initially, we examined allelic loss at five markers flanking DICE1 in 56 ESCC patients from Shanxi Province, China, and then examined the entire coding sequence of this gene for mutations using polymerase chain reaction-single-strand confirmation polymorphism (PCR-SSCP) analysis and DNA sequencing. Subsequently, we extended our evaluation to an additional 80 ESCC patients and 232 healthy individuals to confirm the germline variant found in the initial 56 ESCC patients. The frequencies of allelic loss were 71, 58, and 75% for D13S325, D13S757, and D13S887, respectively, in the initial 56 ESCC patients studied. Overall, 73% of informative patients had loss of heterozygosity (LOH) for at least one of these three markers. Somatic mutations were identified in three patients (3/56, 5%), and one novel polymorphism was identified in 3% of ESCC patients (4/136) and 3% of healthy individuals (6/232). We conclude that DICE1 mutations occur in ESCC but are infrequent. The candidate tumor suppressor gene corresponding to the frequent allelic loss on chromosome 13q14 in ESCC remains unknown.     

MicroRNA-34b has an oncogenic role in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is a common malignancy and one of the more difficult diseases to diagnose in Japan due to its poor prognosis. MicroRNAs are small non-coding RNAs of 21-23 nucleotides that regulate gene expression. MicroRNA-34b (miR-34b) has been reported to be overexpressed in various types of cancer. However, its role in ESCC has yet to be extensively studied. The present study investigated the expression of miR-34b in 88 ESCC patients. The miR-34b expression in ESCC was significantly higher than that in the corresponding normal esophageal mucosa. It was more highly expressed in tumors with more advanced stages. However, its expression did not correlate with the p53 status. Transfection of anti-miR-34b to the ESCC cells suppressed cell growth in vitro. These results suggest an oncogenic role of miR in ESCC.     

Interleukin 13 participates in terminal differentiation of esophageal squamous cell carcinoma cells

BackgroundIn China, esophageal squamous cell carcinoma (ESCC) accounts for more than 90% of all esophageal cancer cases. Interleukin 13 (IL-13) was widely reported to play a key role in tumor progression. Our previous study reported that IL-13 was a favorable predictive marker for the overall survival of esophageal squamous cell carcinoma (ESCC) patients, but how IL-13 contributes to ESCC progression remains unknown. This study aims to explore the role of IL-13 and its underlying downstream molecular mechanisms in ESCC progression.MethodsTissue microarrays including 262 primary ESCC tumor tissues were collected and analyzed. The expression of IL-13 in ESCC tumor tissue was detected with immunohistochemistry staining (IHC). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to qualify the expressions of KRT13, KRT4 and 15-lipoxygenase-1 (15-LOX-1) in cultured ESCC cell lines with recombinant IL-13 treatment.ResultsIL-13 was expressed in the esophageal epithelium cells and ESCC tumor cells. High IL-13 expression in ESCC tumor cells predicted a good prognosis for patients. Recombinant human IL-13 raised KRT13 and 15-LOX-1 mRNA levels, but lowered KRT4 mRNA level 15-LOX-1 in ESCC cells in vitro.ConclusionsIn summary, our study suggests that IL-13 might improve the prognosis of ESCC by promoting the terminal differentiation of ESCC cells. This may offer potential new therapeutic target for early treatment of ESCC.     

Combination effects of distinct cores in 11q13 amplification region on cervical lymph node metastasis of oral squamous cell carcinoma

Lymph node metastasis (LNM) in oral squamous cell carcinoma (OSCC) is known to associate with a significant decrease of 5-year survival. Genetic factors related to the difference of the LNM status in the OSCC have been not fully elucidated. Array-based comparative genomic hybridization (CGH) with individual gene-level resolution and real-time quantitative polymerase chain reaction (QPCR) were conducted using primary tumor materials resected from 54 OSCC patients with (n=22) or without (n=32) cervical LNM. Frequent gain was observed at the 11q13 region exclusively in patients with cervical LNM, which was confirmed by real-time QPCR experiments using 11 genes (TPCN2, MYEOV, CCND1, ORAOV1, FGF4, TMEM16A, FADD, PPFIA1, CTTN, SHANK2 and DHCR7) in this region. It was revealed that two distinct amplification cores existed, which were separated by a breakpoint between MYEOV and CCND1 in the 11q13 region. The combination of copy number amplification at CTTN (core 2) and/or TPCN2/MYEOV (core 1), selected from each core, was most significantly associated with cervical LNM (P=0.0035). Two amplification cores at the 11q13 region may have biological impacts on OSCC cells to spread from the primary site to local lymph nodes. Further study of a larger patient series should be conducted to validate these results.     

A novel gene,NMES1, downregulated in human esophageal squamous cell carcinoma

To isolate genes with different expression levels in human esophageal SCC, SSH and reverse Northern were performed between cancer tissue and its normal counterpart. Among the differentially expressed genes identified, we report here cDNA corresponding to a 0.88 kb mRNA (NMES1), whose expression was observed in all 36 adjacent normal esophageal mucosae, while 31 (86%) matched cancer tissues showed a marked reduction or complete lack of its expression. Sequence analysis of its full-length cDNA revealed a gene encoding a predicted polypeptide of 9 kDa. Northern blot showed that NMES1 was distributed mainly in the alimentary tract. The gene was mapped to 15q21.1 by screening the GenBridge 4 RH panel. Immunohistochemistry confirmed the downregulation of NMES1 in esophageal SCC at the protein level and showed that it is a nuclear protein. In situ hybridization revealed its different expression during mouse embryonic development, especially in bone, brain, stomach and intestine. The negative correlation of NMES1 expression with esophageal oncogenesis suggests its suppressive role in tumorigenesis of the esophagus, while the precise function of NMES1 still needs further investigation.     

Frequent amplification of chromosomal region 20q12-q13 in ovarian cancer.

DNA amplification at chromosomal region 20q12-q13, which is common in breast cancer, has recently been described also in ovarian tumors. We studied the amplification of the recently identified candidate oncogenes in this region in 24 sporadic, 3 familial and 4 hereditary ovarian carcinomas, and in 8 ovarian cancer cell lines. High-level amplification of at least one of the five nonsyntenic regions at 20q12-q13.2 was found in 13 sporadic (54%) and in all four hereditary tumors. Typically, two or more distinct amplicons (separated by nonamplified DNA) were found coamplified in various combinations. The regions defined by the AIB1 and PTPN1 genes (at 20q12 and 20q13.1, respectively) were amplified in 25% and 29% of the sporadic tumors, also without simultaneous coamplification of other regions. Amplification of AIB1 (a steroid receptor coactivator gene) was associated with estrogen receptor positivity in sporadic ovarian carcinomas (P = 0.01) and showed a tendency to correlate with poor survival of patients. Of the genes amplified in breast cancer, the BTAK gene was amplified in 21%, the MYBL2 gene in 17%, and the ZNF217 gene in 12.5% of the sporadic tumors. The high frequency of gene amplification at 20q12-q13.2 suggests that the genes amplified therein may play a central role in the pathogenesis of sporadic and hereditary ovarian carcinoma.     

A comparative analysis of clinicopathological factors and survival between esophageal basaloid squamous cell carcinoma and conventional esophageal squamous cell carcinoma

IntroductionRecently, the number of diagnosed esophageal basaloid squamous cell carcinoma (EBSCC) has gradually increased. However, available data on EBSCC are limited to date.MethodsA total of 165 EBSCC (Cohort 1) and 515 conventional esophageal squamous cell carcinoma (ESCC) (Cohort 2) were retrospectively analyzed.ResultsIn Cohort 1, 70 cases only had invasive EBSCC component (42.4%, defined as Group 1), 73 cases had concomitant invasive ESCC component (44.2%, Group 2), and 22 had concomitant invasive poor-differentiated component (13.3%, Group 3). Lymph node metastasis rates of Group 3, Group 2 and Group 1 were ranked from high to low (P = 0.044). There were higher patient age (P = 0.047), smaller tumor size (P = 0.009), more nerve invasion (P < 0.001), and lower pTNM stage (P < 0.001) in EBSCC (Cohort 1), compared with ESCC (Cohort 2). In Cohort 1 and Cohort 2, pTNM stage was an independent prognostic factor for both DFS and OS. No significant survival difference was found between EBSCC (Cohort 1) and ESCC (Cohort 2) in pIA-B stage, pIIA-B stage, pIIIA-B stage and pIVA-B stage (P > 0.05).ConclusionOur analysis of the largest EBSCC series from a single institution to date with conventional ESCC demonstrated that EBSCC carried a similar prognosis with ESCC in pIA-B stage, pIIA-B stage, pIIIA-B stage and pIVA-B stage. And pure EBSCC, didn't have poorer survival than mixed EBSCC with concomitant ESCC or other components. Our findings may be valuable in the better understanding of EBSCC's biological behaviors, and the related molecular mechanism is needed to be explored in the future.     

食管癌肿瘤条件培养新生血管形成机制的探讨

目的:探讨Survivin、Survivin-△Ex3及VEGF在食管癌细胞诱导血管形成中作用的相关性.方法:用食管鳞癌肿瘤条件培养基培养人脐静脉内皮细胞,用ELISA法检测肿瘤条件培养基中VEGF蛋白的表达情况.用RT-PCR法检测内皮细胞中Survivin、Survivin-△Ex3及VEGF的表达情况.结果:肿瘤条件培养基中VEGF蛋白表达随培养时间呈上升趋势.实验组人脐静脉内皮细胞中Survivin在更换肿瘤条件培养基后4及10 h时表达明显增加,与对照组比较差异有统计学意义(CMvsRPMI:4 h:1.019 7±0.058 8 vs 0.658 8±0.043 7,P=0.000 5;10 h:0.797 1±0.074 4 vs0.444 9±0.041 2,P=0.001 0),而Survivin-△Ex3在实验组和对照组间的表达差异无统计学意义(CM vs RP-MI:4 h:0.423 7±0.087 0 vs 0.294 0±0.043 1,P=0.109 1;10 h:0.372 7±0.084 8 vs 0.226 4±0.009 2,P=0.078 1),VEGF在4及10 h时表达明显下降,与对照组比较差异有统计学意义(CMvsRPMI:4 h:0.276 7±0.065 8 vs 0.977 8±0.044 5,P=0.000 0;10 h:0.173 9±0.024 1 vs 0.917 8±0.124 7,P=0.004 8).结论:食管癌肿瘤条件培养基可通过VEGF作用于人脐静脉内皮细胞表面的受体,进而增加人脐静脉内皮细胞中Survivin的表达,促进肿瘤新生血管形成,同时抑制血管内皮细胞中VEGF的表达,这为食管癌肿瘤血管形成的发生机制及防治提供一定的实验依据.     

Expression analysis of genes at 3q26-q27 involved in frequent amplification in squamous cell lung carcinoma

Gene amplifications are known to occur frequently in lung cancer. Recently, we identified gene amplifications at 3q26 in squamous cell lung carcinoma (SCC) using reverse chromosome painting. Here, our aim was to analyse the expression of genes which map within the amplified chromosomal region. The genes which were selected for their known function and their potential involvement in tumour development included the genes for ribosomal protein L22 (RPL22), butyrylcholinesterase (BCHE), glucose transporter 2 (SLC2A2), transferrin receptor (TFRC), thrombopoietin (THPO) and the phosphatidylinositol-3 kinase catalytic alpha polypeptide (PIK3CA). While five genes were expressed in the majority of the 17 samples of SCC, the gene for the glucose transporter 2 (SLC2A2) was expressed in only three cases, excluding SLC2A2 as the target gene of the amplification unit. For a subset of tumours, we determined the amplification status of the six genes. The TFRC, PIK3CA, BCHE, THPO and SLC2A2 genes were amplified in several cases, whereas the RPL22 gene was amplified in only one case. The combined amplification and expression data of this and our previous studies indicate that the amplified region at 3q26 contains several genes that are transcribed in SCC, providing the possibility that several amplified and functionally important genes at 3q26 may be involved in the pathogenesis of SCC.     

微小 RNA 与食管鳞状细胞癌

微小 RNA（miRNA）可通过细胞信号转导、上皮间质转化、血管生成等调控机制影响食管鳞状细胞癌细胞的增殖、凋亡、侵袭和转移。特定的血清 miRNA 可作为食管鳞状细胞癌诊断、预后的新型肿瘤标志物。近来研究表明 miRNA 可提高食管鳞状细胞癌放疗敏感性甚至逆转多药耐药,具有极大的临床价值。     

Quantitative tissue proteomics of esophageal squamous cell carcinoma for novel biomarker discovery

Esophageal squamous cell carcinoma (ESCC) is among the top ten most frequent malignancies worldwide. In this study, our objective was to identify potential biomarkers for ESCC through a quantitative proteomic approach using the isobaric tags for relative and absolute quantitation (iTRAQ) approach. We compared the protein expression profiles of ESCC tumor tissues with the corresponding adjacent normal tissue from ten patients. LC-MS/MS analysis of strong cation exchange chromatography fractions was carried out on an Accurate Mass QTOF mass spectrometer, which led to the identification of 687 proteins. In all, 257 proteins were identified as differentially expressed in ESCC as compared to normal. We found several previously known protein biomarkers to be upregulated in ESCC including thrombospondin 1 (THBS1), periostin 1 (POSTN) and heat shock 70 kDa protein 9 (HSPA9) confirming the validity of our approach. In addition, several novel proteins that had not been reported previously were identified in our screen. These novel biomarker candidates included prosaposin (PSAP), plectin 1 (PLEC1) and protein disulfide isomerase A 4 (PDIA4) that were further validated to be overexpressed by immunohistochemical labeling using tissue microarrays. The success of our study shows that this mass spectrometric strategy can be applied to cancers in general to develop a panel of candidate biomarkers, which can then be validated by other techniques.